In between beats, relaxation of the heart is crucial to maintain contractile function. Without adequate relaxation, insufficient filling will result in inadequate blood supply for the organism. In order to ultimately treat, or even prevent diastolic dysfunction, we need to understand the basic process of cardiac relaxation. Although relaxation is often thought of as a merely passive process that follows contraction, this is far from the truth. The relaxation process is the final physiological parameter of the sum of (inter) actions of calcium removal from the cytosol on one hand, and myofilament properties on the other. To date, it remains unresolved how, and under which conditions, these two factors govern the cardiac relaxation process in large mammals. To increase our understanding of the processes involved in cardiac relaxation, we propose to test the hypothesis that under physiological conditions, myofilament properties dictate the rate of relaxation in adult mammalian myocardium. To test this hypothesis, we will employ the following aims: 1) Measure calibrated intracellular calcium transients in ultra-thin rabbit cardiac trabeculae to study the coupling between calcium and force decline. This model will deliver results that can be well extrapolated to human physiology because of similarities in calcium handling and myofilament composition, unlike mice or rats, whose properties are remote from human. 2) Evaluate the influence of sarcoplasmic reticulum (SR) function on the governing of the relaxation phase at different levels of SR load and impaired function. 3) Evaluate the influence of preload on the governing of the relaxation phase by assessment at different sarcomere length. 4) Test the proof-of-principle that single molecular alterations can change the speed of relaxation. We will use adenoviral-mediated expression of the fast SR calcium ATP-ase pump (Serca1a) to speed up relaxation and a cardiac troponin-C mutant (161QcTnC; with lowered calcium affinity) to shorten myofilament-governed relaxation. Our long-term goal towards understanding, treating, and curing cardiac relaxation disorders can only be achieved if we understand the normal governing of cardiac relaxation. Results of the proposal will provide groundlaying information as to how cardiac relaxation is governed, and should aid in devising hypothesis driven strategies to combat diseases in which the relaxation process is disturbed in the future.